Neoplaether, a new cytotoxic and antifungal endophyte metabolite from Neoplaconema napellum IFB-E016

A new diphenyl ether, named neoplaether, together with five known compounds monomethylsulochrin, physcion, helvolic acid, ergosterol and ergosterol peroxide, was isolated from the culture of Neoplaconema napellum IFB-E016, an endophytic fungus residing in the healthy leaves of Hopea hainanensis. The structure of neoplaether was elucidated by a correlative interpretation of its infrared spectrometry, mass spectroscopy and nuclear magnetic resonance spectra, and confirmed by its single-crystal X-ray diffraction analysis. Neoplaether exhibited significant cytotoxic activity against the human nasopharyngeal epidermoid tumor KB cell line, with an IC(50) value of 13.0 microg mL(-1), comparable to that of 5-fluorouracil (2.5 microg mL(-1)) co-assayed as a positive reference. In addition, it showed antifungal activity against Candida albicans, with a minimal inhibitory concentration value of 6.2 microg mL(-1) (amphotericin as a positive control had a value of 1.5 microg mL(-1)).     

PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265–269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.     

Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70-80 degrees C (k(cat)/K(m)=8.3 mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K(m)=667 mM(-1) s(-1)), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.     

Thermostabilization of ovotransferrin by anions for pasteurization of liquid egg white

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T(m), causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T(m)(DSC), was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T(m) of oTf by about 5-7 degrees C. However, neither sodium chloride nor sodium bicarbonate raised T(m) by that much. Temperature T(m) was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T(m)(DSC), suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.The Van't Hoff plot from the T(m) and DeltaH(d) values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T(m).     

Characterization of mitochondrial iron uptake in HepG2 cells

There is increasing evidence that accumulation of redox-active iron in mitochondria leads to oxidative damage and contributes to various neurodegenerative diseases, such as Friedreich's ataxia and Parkinsons disease. In this work, we examined the existence of regulatory mechanisms for mitochondrial iron uptake and storage. To that end, we used rhodamine B-[(1,10-phenanthrolin-5-yl)amino carbonyl] benzyl ester, a new fluorescent iron-sensitive probe that is targeted specifically to the mitochondrion. We found that extracellular iron was incorporated readily into mitochondria in an apparently saturable process. Moreover, the rate of iron incorporation responded to the Fe status of the cell, an indication that the mitochondrion actively regulates its iron content.     

Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation

We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.     

In vitro cytotoxicity of norditerpenoid alkaloids

Forty-three norditerpenoid alkaloids isolated from Aconitum, Delphinium and Consolida species have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma), SkMel25 (human melanoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the non-tumor cell line CHO (Chinese hamster ovary cells). Neoline (5), 8-O-methylcolumbianine (6), 1,14-diacetylcardiopetaline (9), 18-O-demethylpubescenine (13), 14-deacetylpubescenine (14), pubescenine (15), 14-deacetylajadine (25), lycoctonine (26), browniine (28), delphatine (29), dehydrotakaosamine (34), and ajadelphinine (37) exhibited selective cytotoxicity to cancerous versus non-cancerous cells. Some of these compounds had an irreversible effect on SW480 (5, 15, 25, 26, and 34), HeLa (15, 34, and 37) and SkMel25 (15 and 34) cell lines. In order to gain insights into the mechanism of irreversible cytotoxic action of these compounds we compared the cell viability by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and the acid phosphatase (AP) methods. Our results suggest that the effects of these compounds could be related to the inhibition of ATP production.     

T-antigen of the human polyomavirus JC attenuates faithful DNA repair by forcing nuclear interaction between IRS-1 and Rad51

JC polyomavirus (JCV), which infects 90% of the human population, is detectable in human tumors. Its early protein, JCV T-antigen, transforms cells in vitro and is tumorigenic in experimental animals. Although T-antigen-mediated transformation involves genetic alterations of the affected cells, the mechanism underlying this genomic instability is not known. We show that JCV T-antigen inhibits homologous recombination DNA repair (HRR), which results in an accumulation of mutations. T-antigen does not operate directly but utilizes a cytosolic molecule, insulin receptor substrate 1 (IRS-1). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with artificial nuclear localization signal. Our observations define a new mechanism by which viral protein utilizes cytosolic molecule to inhibit faithful DNA repair, and suggest how polyomaviruses could compromise stability of the genome. (c) 2005 Wiley-Liss, Inc.